tbuckley/cabot-search huggingface dataset:
Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction
Authors: Kaya Florczak, Aizhu Lu, Jerry Wang, Ruonan Gu, Ying Xia, Hongwei Wang, Darryl R Davis, Wenbin Liang
Journal: Circulation Research (2023)

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p&lt;0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p&lt;0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p&lt;0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p&lt;0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p&lt;0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.
Abstract P3073: Interleukin-2 Receptor Gamma Signaling Is Required For The Regenerative Adaptive Immune Response In The Adult Zebrafish Heart
Authors: Qianchen Wang, João Cardeira-da-Silva, Didier Stainier
Journal: Circulation Research (2023)

Ischemic heart disease remains a leading cause of death worldwide. Unlike adult mammals, zebrafish have the ability to fully regenerate their heart after injury through processes including epicardium and endocardium activation, coronary revascularization, cardiomyocyte dedifferentiation and proliferation, and scar resolution. Immune cells are critical to remove damaged tissue, sustain repair and initiate regeneration after injury. Our previous data show that blocking antigen presentation impairs cardiac regeneration, pointing at a role for the adaptive immune system during this process. However, the molecular mechanisms underlying a pro-regenerative adaptive immune response remain largely unknown. Based on bulk transcriptomics, single-cell transcriptomics, and qPCR data, we have identified il2rga ( interleukin-2 receptor gamma a ) and jak3 ( janus kinase 3) as potential regulators of the adaptive immune response during cardiac regeneration. We indeed found that il2rga mutants exhibit a reduced expression of T-cell markers, as well as impaired cardiac regeneration, as observed by decreased cardiomyocyte dedifferentiation and proliferation, and defective scar resolution. In addition, il2rga mutants display decreased expression of pro-regenerative factors (e.g., igf1, ngfb, nrg1, pdgfaa, tgfb2, vegfc ). Similarly, loss of Jak3, which functions downstream of Il2rga, also leads to reduced cardiomyocyte dedifferentiation and proliferation, as well as impaired endocardium activation. Bulk transcriptomics further shows the downregulation of key genes involved in the adaptive immune response in il2rga mutants. Specifically, we found that il13 , a recently described cardiomyocyte cell cycle regulator and T helper 2 (Th2) cytokine, is strongly downregulated upon the loss of il2rga . Altogether, our findings reveal the crucial role of Il2rga/Jak3 signaling during cardiac regeneration, thereby opening new avenues to identify and understand the adaptive immune mechanisms capable of sustaining regenerative responses post-cardiac injury.
Abstract P3170: Immunomodulatory Effects Of Exosomes From Epigenetic Drugs-Stimulated Macrophages
Authors: Selene Perales, Vinoth Sigamani, Sheeja Rajasingh, Rajasingh Johnson
Journal: Circulation Research (2023)

Background: Exosomes have significant therapeutic promise; however, a key problem prohibiting and exploring exosomes in clinical therapy is the availability of disease-specific exosomes. Our recent in vitro and in vivo studies identified that combined treatment with Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli. Here, we elucidate the therapeutic potential of exosomes derived from the macrophages treated with P+T during the inflammatory condition. Hypothesis: We hypothesize that exosomes derived from LPS-challenged macrophages (THP1) treated with P+T will provide a better anti-inflammatory response than direct drug treatment. Methods and Results: Macrophages were grown in exosome-depleted media for 72 hrs and stimulated once with LPS, P+T, or L+P+T for exosome collection. These exosomes were used for treating LPS-challenged macrophages. qRT-PCR analysis was performed to check for the expression of pro-inflammatory genes after exosome treatment. Our results show that LPS-challenged macrophages treated with exosome from L+P+T showed significantly reduced expression of TNFα and IL1β when compared to the direct drug treatments (Fig. A, B) . We also observed that the pro-inflammatory microRNAs such as miR-21 and miR181-a were significantly reduced in the L+P+T stimulated exosome group (Fig. C, D) . Conclusions: We show for the first time that drug-stimulated exosomes are more effective in reducing inflammation than direct drug treatment. These specific exosomes could be used for infection-related therapy.
Abstract P3029: The Role Of Fatty Acid Synthase In The Modulation Of Vascular Smooth Muscle Cells Into A Foam Cell Phenotype
Authors: Bethany J Bogan, Holly C Williams, Claire Holden, Amir Rezvan, Alejandra San Martin
Journal: Circulation Research (2023)

Background: The contractile phenotype of differentiated VSMCs is essential for vascular function. When exposed to Cholesterol (CHO), VSMCs dedifferentiate and acquire characteristics of foam cells, lipid-laden macrophage-like cells that comprise the Atherosclerotic plaques. CHO uptake by VSMCs is either removed by ABCA1-mediated efflux or is stored in lipid droplets as cholesterol esters (CEs). CEs are formed by the reaction of CHO with fatty acids catalyzed by SOAT1. Thus, we hypothesize that fatty acid biosynthesis controls the formation of VSMC-derived foam cells. Results: In VSMCs, CHO treatment upregulates the lipogenic enzymes FASN (3.6±1 vs. 9.0±0.9 AU p&lt;0.01) and acetyl-CoA carboxylase (ACC) (2.8±0.5 vs. 10.2±2.3 AU p=0.02) and induces the phosphorylation of ATP Citrate Lyase (ACLY) (1.4±0 vs. 6.2±1 AU). Consistent with the literature, these changes are accompanied by increased expression of the macrophage-related markers CD68 and KLF4. Significantly, downregulation of FASN inhibits the CHO-induced CD68 (9.1±0.8 vs. 4.6±1,9 p=0.002) and KLF4 (p=0.02) expression upregulation. FASN-deficient VSMCs failed to accumulate lipids and transition to a foam cell phenotype after CHO treatment measured by Oil Red O staining. Interestingly, FASN-deficient VSMCs have decreased expression of SOAT1 (p=0.04) and increased expression of the CHO efflux transporter ABCA1 (p=0.04). Conclusion: Our study shows lipogenic enzymes are upregulated during the VSMC to foam cell transition triggered by CHO treatment. FASN expression is required for VSMC-derived foam cell formation. Our data suggest that FASN participates in the esterification of CHO and foam cell formation and may contribute to atherosclerotic plaque accumulation. Thus, FASN may represent a novel target for therapeutic intervention.
Abstract P2030: Regulation Of Pathologic Cardiac Signaling Via Rac1 Palmitoylation
Authors: James P Teuber, Arasakumar Subramani, Sneha Dinda, Matthew Brody
Journal: Circulation Research (2023)

Development of more effective therapies to prevent maladaptive cardiac remodeling and heart failure would be greatly aided by the identification of novel signaling pathways that regulate cardiac hypertrophy. Cysteine palmitoylation is a reversible post-translational lipid modification that dynamically regulates intracellular trafficking and subcellular compartmentalization of signaling proteins. Previous work from our group identified that mice with cardiomyocyte (CM)-specific overexpression of zDHHC3, a Golgi-localized S-acyltransferase, develop lethal dilated cardiomyopathy. S-acyl-proteomics revealed Cys-178 of Rac1, a small GTPase, as a palmitoylation site modified by zDHHC3 and hearts from zDHHC3-overexpressing mice display elevated levels of palmitoylated, active, and total Rac1, suggesting Rac1 palmitoylation promotes its activation and may drive pathologic cardiac remodeling. Rac1-dependent activation of NADPH oxidase 2 (Nox2) in CMs is required for angiotensin II (AngII)-induced cardiac hypertrophy and myocardial oxidative stress, suggesting inhibition of Rac1/Nox2 signaling could potentially ameliorate adverse cardiac remodeling. We found Rac1 palmitoylation at Cys-178 to be required for hypertrophy and oxidative stress in neonatal rat CMs as reactive oxygen species production and hypertrophy elicited by expression of a constitutively active Rac1 mutant were attenuated by mutation of the Cys-178 palmitoylation site, suggesting palmitoylation targets Rac1 to the Nox2 complex to promote myocardial oxidative stress. Notably, Rac1 palmitoylation and oxidative stress were also enhanced in hearts of mice subjected to AngII infusion and transgenic mice with CM-specific overexpression of the angiotensin II receptor type I (AT1R), suggesting a role for Rac1 palmitoylation in AngII-induced maladaptive cardiac signaling. To further elucidate the role of Rac1 palmitoylation in cardiac pathology, we developed a CM-specific knock-in mouse model in which Cys-178 of Rac1 is mutated to serine. Ongoing work utilizing this model will investigate mechanisms by which palmitoylated and depalmitoylated Rac1 differentially regulate signaling, oxidative stress, and hypertrophy in CMs in response to pathologic stimuli.
Abstract P2022: Ankyrin-R Isoforms Are Differentially Expressed In Cardiac Tissues Under Stress
Authors: Aaron D Argall, Holly Sucharski-Argall, Luke Comisford, Casey Crawford, Thomas J Hund, Peter J Mohler, Sara N Koenig
Journal: Circulation Research (2023)

Heart disease remains the leading cause of death in the United States with heart failure specifically accounting for 13.4% of all heart disease related deaths. In 2019, Andersson et al., utilized a multi-omics approach to evaluate participant samples from the Framingham Heart Study and showed that ankyrin-R (AnkR; encoded by ANK1 ) is significantly associated with diastolic function, left ventricular remodeling and heart failure with preserved ejection fraction. Ankyrins are a family of proteins that link integral membrane proteins with the actin/β-spectrin cytoskeleton. Ankyrins-B/G have been extensively studied and identified within the heart and their dysfunction is associated with cardiac structural and electrical phenotypes. Ankyrin-R was first identified in red blood cells and has yet to be studied in the context of cardiac function and heart failure or arrhythmia disease. To study AnkR in the context of the heart we isolated perfused tissues from adult wild-type mice and performed immunoblot and qPCR analysis on ankyrin-R protein and Ank1 mRNA expression. The large AnkR isoform is expressed in the heart, along with the brain, intestine, and spleen. Interestingly, only the heart showed expression of a small AnkR isoform that has previously been shown to interact with the sarcoplasmic protein obscurin. Notably, isolated cardiomyocytes express the small AnkR isoform while cardiac fibroblasts express the canonical large AnkR isoform at both the protein and mRNA level. Canonical AnkR is diffusely expressed in the fibroblast membrane, cytoplasm, cytoskeleton, and soluble nuclear fractions. Interestingly, this expression pattern is switched under stressed conditions where cardiomyocytes increase and cardiac fibroblasts decrease their canonical AnkR expression, respectively. These preliminary results are the first to show canonical AnkR expression in the mouse myocardium specifically within the cardiac fibroblasts and allude that AnkR’s utilization during cardiac stress is cell-type dependent. Future studies will seek to define molecular, cell, and organ phenotypes related to AnkR in the heart.
Abstract P3064: Cytotoxic T-cells Drive Doxorubicin Induced Cardiac Fibrosis And Systolic Dysfunction
Authors: Abraham L Bayer, Maria Zambrano, Hannah Elwell, Sasha Smolgovsky, Kuljeet kaur, Aarti Asnani, Pilar Alcaide
Journal: Circulation Research (2023)

Background: Doxorubicin (DR), the most commonly prescribed chemotherapy, results in dose-dependent cardiotoxicity and heart failure (HF), and patients respond less frequently to standard HF treatments. DR causes oxidative stress and myocyte death, both potent inducers of a T-cell immune response and cardiac inflammation in other causes of HF. However, mechanistic depth on the immune response elicited by DR is limited. We hypothesized that an aberrant T-cell immune response secondary to cardiac damage by DR contributes to DR-induced HF and cardiac remodeling. Methods and Results: We treated wild type (WT) and T-cell deficient ( Tcra -/- ) mice with 5.0 mg/kg DR or PBS weekly by intraperitoneal injection for 4 or 8 weeks. We found at both time points Tcra -/- mice had preserved ejection fraction (EF) by echocardiography and decreased cardiac fibrosis by picrosirius red staining, compared to WT. At 8 weeks, Tcra -/- mice had fewer TUNEL + cardiac cells than WT mice. DR treated mice had increased frequency of activated effector CD8 + CD62L lo CD44 hi cells in the cardiac draining lymph nodes, increased circulating CD8 + T-cells, and cardiac CD8 + T-cell infiltration at both time points. Histologic analysis demonstrated localization of CD8 + T-cells adjacent to cardiac fibroblasts (CFB) in the hearts of DR treated mice. Targeted antibody depletion of CD8 + but not CD4 + cells prior to 4 week DR treatment resulted in improved EF and decreased cardiac fibrosis. Moreover, cardiac CD8 + T-cells expressed higher IFNγ in vivo in DR compared to PBS treated mice, and DR hearts had increased gene expression of the IFNγ inducible chemokines Cxcl9 and Cxcl10 . Ex vivo DR (0.1 μg/mL) treatment of CD8 + T-cells during TCR activation with αCD3/CD28 increased IFNγ expression at the protein and transcript level. The same dose of DR also increased surface expression of ICAM-1 on CFB, and CD8 + T-cells adhered to DR treated CFB in vitro in greater numbers than untreated CFB, and induced more αSMA expression. Lastly, in patients treated with DR, plasma levels of CXCL9 and CXCL10 correlated with decreases in EF after 3 months of treatment. Conclusion: These data outline a novel CD8 T-cell-driven inflammatory mechanism in DR cardiotoxicity, driven by IFNγ-inducible chemokines in animal models and patients.
Abstract P1098: Molecular Maturation Of Engrafted Human Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Juliana Gomez, Wenlei Jiang, Beiping Qiang, Gary Bader, Michael A Laflamme
Journal: Circulation Research (2023)

The transplantation of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) represents an attractive strategy to regenerate myocardium lost following injury and mitigate the pathological remodeling that ultimately leads to heart failure. However, in vitro hPSC-CMs have an immature phenotype that likely contributes to the transient arrhythmias observed during the initial weeks following hPSC-CM transplantation in large-animal models, hindering safe translation into the clinic. We hypothesized that maturational changes in hPSC-CMs in vivo facilitate the improved electrophysiological behavior of graft tissue observed at later time-points. Because the changing phenotype of hPSC-CM graft tissue remains largely unexplored, we used single nucleus RNA sequencing to evaluate the transcriptional dynamics and cell population heterogeneity of hPSC-CMs prior to transplantation and at 2- and 8-weeks following transplantation in a guinea pig cardiac cryoinjury model (n=3). We used methods based on the sorting of nuclei labeled by a genetically-encoded fluorescent reporter to enrich the proportion of human nuclei sequenced from the graft site and created a comprehensive atlas (&amp;gt; 20,000 cells) of host and graft cells present at the implantation site at both time-points. We identified an upregulation of genes involved in intracellular calcium handling, AMPK, and cAMP signaling pathways, as well as a metabolic shift in engrafted hPSC-CMs that we speculate supports their growth and enhanced electrophysiological function. Our study includes a comprehensive description of the temporal changes in hPSC-CM graft tissue at the single-cell resolution and provides unique insights into how these cells adapt to the injured heart environment and mature in vivo.
Abstract GS.13: Dworf Overexpression In The Heart Results In Enhanced Mitochondrial Function
Authors: Omar Brito-Estrada, Cat A Makarewich, Aaron Gibson
Journal: Circulation Research (2023)

Objective: Previously, we identified dwarf open reading frame (DWORF) as a microprotein that regulates SERCA2a in the sarcoplasmic reticulum (SR). New data from our lab indicates that a fraction of DWORF localizes to the mitochondria. The goal of these studies was to analyze the role of DWORF in the mitochondria. Methods &amp; Results: To identify novel DWORF interacting proteins, we performed a BioID assay in neonatal rat ventricular myocytes (NRVMs) expressing a mTurbo-DWORF fusion protein. In addition to identifying SR protein SERCA2a, we unexpectedly identified several mitochondrial proteins. Percoll gradient fractionation of mouse heart lysates showed DWORF is distributed across SR, mitochondria associated membranes (MAMs), and purified mitochondrial fractions. Seahorse mito stress test performed in isolated mitochondria from wildtype (WT), DWORF transgenic (Tg), DWORF knockout (KO), and PLN KO mouse hearts showed that mitochondria from DWORF Tg mice have increased maximal O 2 consumption rates (OCR) while mitochondria from DWORF KO hearts have reduced OCR compared to WT. To measure mitochondrial Ca 2+ dynamics, we performed mitochondrial Ca 2+ uptake assays and found that DWORF Tg hearts have increased Ca 2+ uptake rates while DWORF KO mice have reduced Ca 2+ uptake rates compared to WT. Western blot (WB) analysis of mito extracts from WT and DWORF Tg hearts indicated elevated levels of the mitochondrial Ca 2+ uniporter (MCU), while no observable changes were detected in electron transport chain (ETC) subunit expression. WB analysis also revealed a reduction in phosphorylated pyruvate dehydrogenase (PDH) in DWORF Tg mice, indicating that the enhanced mitochondrial activity observed in DWORF Tg mice may be due to a more active TCA cycle. Additionally, metabolomics analysis shows that DWORF Tg mice have lower levels of fatty acids and carnitines than WT. Conclusions: DWORF overexpression leads to increased mitochondrial respiration, enhanced mitochondrial Ca 2+ uptake, and reduced PDH phosphorylation (increased PDH activity), while DWORF loss-of-function results in the opposite phenotype. Collectively, these data indicate that DWORF plays a previously unrecognized role in the mitochondria and contributes to mito Ca 2+ regulation and energetics.
Abstract P3088: Hexokinase 3 Regulates Macrophage Polarization And Cardiac Remodeling Likely Through Serine Metabolism
Authors: Amir Mahmoodzadeh, Mohammad Keykhaei, Ashley Akrami, Spencer Spencer Duncan, Hossein Ardehali
Journal: Circulation Research (2023)

Myocardial infarction (MI) is associated with activation of macrophages in the myocardium. Hexokinase 3 (HK3) is the mainly expressed hexokinase in macrophage. Along with its role as the first enzyme in glycolysis, HK3 has shown to be involved in the regulation of diverse immune system activities. In this study we studied the role of HK3 in the regulation of macrophage polarization into inflammatory phenotype (termed as M1) and anti-inflammatory and repair phenotype (termed as M2). Using CRISPR-Cas9 technology, we generated mice with deletion of exons 2 and 3 of the Hk3 gene, which are needed for the function of the protein. Bone marrow-derived macrophages (BMDM) isolated from Hk3 -/- mice displayed higher levels of M2 markers, including Arg1, Ym1, Mrc1, PD-L2, IRF4, Mgl2, and Mgl1 compared to BMDMs from wild-type mice. Also, we found that pSTAT6 protein, was higher in BMDMs from Hk3 -/- mice. Furthermore, there was a higher oxygen consumption rate and lower extracellular acidification rate in Hk3 -/- BMDMs. Steady-state metabolomics in BMDMs isolated from WT and Hk3 -/- mice showed that there was a significant increase in metabolites in the serine biosynthesis pathway (SBP), indicating that glucose metabolism is more shunted into the SBP with Hk3 deletion. The removal of serine/glycine from media resulted in a significant decrease in the M2 markers in wild-type BMDMs; However, higher M2 markers were observed in Hk3 -/- BMDMs. This result indicated that the SBP is needed for the polarization of macrophages into the M2 subtype, and HK3 mediated M2 polarization is likely through the SBP. We then subjected Hk3 -/- to LAD and assessed cardiac function after seven days using echocardiography. Result demonstrated that both male and female Hk3 -/- mice display improved cardiac function, as assessed by measurement of ejection fraction (EF), fractional shortening (FS), and interventricular septum thickness in diastole (IVSd). Our results demonstrate that HK3 is a novel regulator of macrophage polarization into the phenotype with more anti-inflammatory and tissue repair characteristic. This study is clinically important by providing a potent novel approach to the treatment of ischemic heart disease.